Targeted gene modification is a powerful tool for researchers interested in functional analysis of genes and potentially for gene therapy applications. The primary limitation of gene targeting is the low frequency with which it occurs in many organisms and cell types, including mammalian cells, occurring in roughly one cell for every 105-107 treated cells (Vasquez, et al., Proc. Natl Acad. Sci. USA, 98:8403-8410 (2001)). The low frequency of gene targeting, which relies on HR is due in part to the much higher frequency of random integration via nonhomologous end joining (NHEJ), which occurs in about 1 cell for every 102-104 treated cells (Vasquez, et al., Proc. Natl Acad. Sci. USA, 98:8403-8410 (2001)).
Several strategies have been used to increase the frequency of gene targeting. It was shown that a DNA double-strand break (DSB) at the target site increases the frequency of gene targeting several orders of magnitude in bacteria (Nussbaum, et al., Genetics, 130:37-49 (1992)), yeast (Storici, et al., Proc. Natl Acad. Sci. USA, 100:14994-14999 (2003)), plants (Puchta, et al., Nucleic Acids Res., 21:5034-5040 (1993)), fruit flies (Banga, et al., Proc. Natl Acad. Sci. USA, 89:1735-1739 (1992)), mice (Rouet, et al., Proc. Natl Acad. Sci. USA, 91:6064-6068 (1994)), human embryonic stem cells (Smith, et al., Nucleic Acids Res., 23:5012-5019 (1995)) and many other cell types. Another strategy to increase gene targeting in mammalian cells has been achieved through the overexpression of key recombination proteins from HR proficient organisms. Overexpression of bacterial RecA led to a 10-fold increase in gene targeting in mouse cells (Schcherbakova, et al., Mutat. Res., 459:65-71 (2000)); likewise, overexpression of yeast Rad52 led to a 37-fold increase in gene targeting in human cells (Di Primio, et al., Nucleic Acids Res., 33:4639-4648 (2005)). Conversely, another approach for increasing gene targeting in human cells involves decreasing the amount of DSB repair through the pathway of NHEJ. In mouse embryonic stem cells, an increase in gene targeting was seen in Ku70 (6-fold), XRCC4 (2-fold) and DNAPK-cs-deficient cell lines (2-fold) (Pierce, et al., Genes Dev., 15:3237-3242 (2001)), and a 3-fold increase in Chinese hamster ovary cells lacking DNAPK-cs (Allen, et al., Proc. Natl Acad. Sci. USA, 99:3758-3763 (2002)). Similarly, knockdown of Ku70 and XRCC4 in human colon cancer cells led to a 30-fold increase in gene targeting (Bertolini, et al., Mol. Biotech., 41:106-114 (2009)). Different from the methodologies mentioned above that focused on increasing HR or decreasing NHEJ, it was shown that knockout of the RAD51 recombinase prevents DSB-induced sister chromatid exchange (Fasullo, et al., Genetics, 158:959-972 (2001)), and thus facilitates gene targeting by single-stranded oligonucleotides at the site of a DSB in both haploid and diploid yeast systems (Storici, et al., Mol. Cell. Biol., 26:7645-7657 (2006)). Gene correction close to a DSB by single-stranded oligonucleotides does not require Rad51, but only the strand annealing function of Rad52 (Storici, et al., Mol. Cell. Biol., 26:7645-7657 (2006)). Thus, deleting Rad51 favours DSB-driven recombination by oligonucleotides by strongly reducing the competition with the sister chromatid and/or the homologous chromosome for DSB repair (Fasullo, et al., Genetics, 158:959-972 (2001); Storici, et al., Mol. Cell. Biol., 26:7645-7657 (2006); Lambert, et al., Oncogene, 20:6627-6631 (2001)). Similarly, it was shown that by knocking down human SMC1, important for HR between sister chromatids, gene targeting increases (Potts, et al., EMBO J., 25:3377-3388 (2006)). Without proximity to the DSB site, the sister chromatid was used less frequently as a donor, shifting repair of the DSB more towards HR with the exogenous donor sequence
Therefore, it is an object of the invention to provide improved compositions and methods for modifying genetic material.
It is another object of the invention to provide methods and compositions for gene therapy.
It is still another object of the invention to provide compositions and methods for targeting oligonucleotides to the specific sites in the genome.